Serine proteases are a group of proteolytic enzymes characterized by having a serine and a histidine residue in their active site. Many well known enzymes belong to this group, for example trypsin, kallikrein, thrombin and plasmin. Several of them have found practical use. Trypsin is used in the leather industry. Thrombin is used as a haemostatic agent to stop bleeding from wounds. Urokinase and tissue plasminogen activator, two other serine proteases, are used clinically as thrombolytic agents in the treatment of acute myocardial infarction. A number of these enzymes have been used extensively as research tools, for instance in protein structure determination. Furthermore, the enzymes are used in various diagnostic kits.
Common to most of the serine proteases are their limited stability in solution. This is mainly caused by autodegradation when left in solution, caused by their property as proteases. This limited stability is a problem when the material has to be stored in solution. Commercial serine protease preparations available today are essentially always in the form of frozen solutions orlyophilized powders, with obvious drawbacks. The extra time needed for dissolution of the powder or thawing of the frozen solution to the correct temperature is the most important issue. There are, however, other problems with these preparations. For frozen solutions, there is a need for controlled temperatures (−20° C.) in all steps from manufacture and transportation to storage. For lyophilized powders, there is a need for a reconstitution solution with an acceptable grade of purity and stability. Also, the material frequently needs to be prepared aseptically (by mixing of the two parts) in an environment which may be non-controlled (such as inclement weather or lack of a clean water supply), and there is a need to verify that the powders have been properly mixed. These are all major drawbacks of the products available today, adding to their complexity of use as well as their cost.
For thrombin, which preferably has to be immediately available for use in arresting bleeding, the stability problems have forced manufacturers to use lyophilized thrombin or deep frozen solutions. These then require a certain amount of time to prepare for use. The two thrombolytic agents urokinase and tissue plasminogen activator are sold in the form of lyophilized preparations that have to be dissolved before use. Since thrombolytic treatment of acute myocardial infarction has to be started as early as possible after onset of the infarction, any time delay caused by such preparation is a problem.
Many efforts have been made to find ways to stabilize the various serine proteases. For trypsin, which degrades itself fairly rapidly, a simple and efficient stabilizing agent is the calcium ion (Sipos T and Merkel J, Biochemistry 9:2766 (1970)). Decreasing the pH to below 4 is also a method that works with some of the enzymes, like trypsin and plasmin, but is not feasible with thrombin, since it is irreversibly inactivated by a pH below 5. Reversible protease inhibitors can be used, but are less popular, since they interfere in a detrimental fashion with the action of the enzyme when they are used by themselves (see below).
For stabilization of tissue plasminogen activator (tPA), addition of the amino acid arginine is conventionally used. The tPA material in clinical use today contains arginine as stabilizer.
Also, a lot of effort has been devoted to find ways to stabilize thrombin solutions. As examples of stabilizing additives, the following proposals may be mentioned: carboxylic acids in high concentrations, EDTA, various amino acids, albumin, polymers such as polyethylene glycol, polyvinyl pyrrolidone and polyvinyl alcohol, glycerol, various inorganic salts, carbohydrates, gelatin, collagen.
Japanese patent application JP2004191367 describes a stabilized thrombin containing test reagent for testing blood coagulation ability. The test reagent contains thrombin and a thrombin inhibitor, and may also comprise one or more thrombin stabilizing compounds selected from calcium ion, an organic acid, a surfactant and a protein.
WO 02/100830, WO 02/22575, WO 00/20394, WO 99/11658, WO 02/37937 and U.S. Pat. No. 5,409,927 all describe different serine protease inhibiting compounds and their use in pharmaceutical compositions for treatment of various disease conditions, such as thrombosis, wherein inhibition of the corresponding serine proteases is indicated.
Nakamura et al. (J. Chrom. A, 1009, (2003), 133-139) describe the use of an immobilized protease inhibitor for affinity chromatography of trypsin-like proteases.
Turner et al. (Biochemistry, 25, (1986), 4929-4935) describe three p-amidinophenyl esters that irreversibly inhibit human factor IXa.
Tsung Fu Yang et al. (Biomacromolecules, 25, (2004), 1926-1932) describe the synthesis of a cationic polymer, N,N-diethylethylenediamin polyurethane, for use in gene delivery.
US patent application 2001/0033837 (corresponding to EP 1 136 084 A1) describes a thrombin preparation containing a non-covalently bound inhibitor as stabilizer. Furthermore, the inhibitor is combined with other stabilizing additives, like sugars or carboxylic acids, which have been previously described in patents or other publications.
JP 2000300250 describes the stabilization of thrombin solutions by addition of polyvinyl alcohol, gelatin or polyvinyl pyrrolidone in different buffer solutions.
In GB 1354761, proteases and amylases are stabilized to various extents by a number of substances, such as aliphatic alcohols, carboxylic acids, heterocyclic compounds containing hydroxyl groups, and aliphatic or alicyclic amines.
Thus, stabilization of a serine protease using inhibitors has been described (for example US 2001/0033837 and JP 2004191367, supra). The problem with this approach is that the inhibitor strongly diminishes the effect of the enzyme, if it is not removed prior to use of the preparation. If a potent inhibitor is used, most of the enzymatic activity is lost. A better approach is to use a reversible inhibitor of intermediate strength. However, even in this case, a considerable part of the initial enzymatic activity will be lost as concentration of the inhibitor is increased in order to get a good stabilization effect.